Vector and method of use for nucleic acid delivery to non-dividing cells

Abstract

A recombinant retrovirus capable of infecting a non-dividing cell and a method of producing such a virus is provided. The recombinant retrovirus is preferably of lentivirus origin and is useful for the treatment of a variety of disorders including neurological disorders and disorders of other non-dividing cells.

What is claimed is: 
     
       1. A method of producing a lentivirus which infects non-dividing cells, said method comprising: a) transfecting a suitable packaging host cell with the following vectors: a first vector providing a nucleic acid encoding a lentiviral gag and a lentiviral pol wherein the gag and pol nucleic acid sequences are operably linked to a heterologous regulatory nucleic acid sequence and wherein the vector is defective for nucleic acid sequence encoding functional ENV protein and wherein the nucleic acid of the first vector is devoid of lentiviral sequences both upstream and downstream from a splice donor site to a gag initiation site of a lentiviral genome;   a second vector providing a nucleic acid encoding a non-lentiviral ENV protein;   a third vector providing a nucleic acid sequence containing a lentiviral packaging signal flanked by lentiviral cis-acting nucleic acid sequences for reverse transcription, packaging and integration; a heterologous nucleic acid sequence, operably linked to a regulatory nucleic acid sequence; and a less than full length gag structural gene; and     b) recovering the recombinant virus.   
     
     
       2. The method of claim 1, wherein the lentivirus is human immunodeficiency virus (HIV). 
     
     
       3. The method of claim 1, wherein the first vector further includes at least a nucleic acid sequence operably linked to a promoter, wherein the nucleic acid sequence encodes a protein selected from the group consisting of VPR, VIF, NEF, VPX, TAT, REV, VPU and any combination thereof. 
     
     
       4. The method of claim 1, wherein the transfecting further includes providing a nucleic acid sequence of a fourth vector, wherein the nucleic acid sequence is operably linked to a promoter and encodes a protein selected from the group consisting of VPR, VIF, NEF, VPX, TAT, REV, VPU and any combination thereof. 
     
     
       5. The method of claim 1, wherein the less than a full length gag structural gene has a closed reading frame. 
     
     
       6. Three lentiviral vectors wherein a first vector comprises a nucleic acid encoding a lentiviral gag and a lentiviral pol wherein the gag and pol nucleic acid sequences are operably linked to a heterologous regulatory nucleic acid sequence and wherein the first vector is defective for nucleic acid sequence encoding functional ENV protein and nucleic acid of first the vector is devoid of lentiviral sequences both upstream and downstream from a splice donor site to a gag initiation site of a lentiviral genome; a second vector providing a nucleic acid encoding a non-lentiviral ENV protein; and a third vector providing a nucleic acid sequence containing a lentiviral packaging signal flanked by lentiviral cis-acting nucleic acid sequences for reverse transcription, packaging and integration; a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and a less than full length gag structural gene, wherein the third vector is devoid of one or more accessory genes, wherein the three vectors, when introduced into a host cell, express lentiviral proteins to form lentiviral virions that are replication defective.